The ultimate goal of this proposal and any subsequent phase II proposal is to develop a unique protein agent to prevent Clostridium difficile associated diseases in those patients at high risk rather than wait to treat their dangerous and costly infections. We aim in this SBIR phase I project to determine the feasibility of oral delivery of a lead, specifically targeted bactericidal protein to eliminate C. difficile cariage without untended collateral damage to the intestinal microbiota. The lead candidate, termed a diffocin, kills 27 of a collection of 28 BI/NAP1/027 strains; and Lawley et al (2010) have described a mouse model of C. difficile carriage. We have recently shown that another engineered R-type bacteriocin administered orally to rabbits can transit the GI tract to act as a specific bactericidal agent killing another bacterial pathogen in the terminal ileum and colon. Thus, after improving the lab scale production of this lead recombinant protein in B. subtilis and conducting preliminary pharmacology and pharmacodynamic studies to guide dosing, we shall evaluate the efficacy of diffocins in C57Bl/6 mice that are asymptomatic carriers and shedders of a sensitive strain of C. difficile. If diffocins eliminate C. difficile from carrier mice, the efect of oral diffocins on the mouse intestinal microbiota will be determined by ribotyping the fecal microbiome of normal and C. difficile carrier mice after diffocin administration.